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Large Molecule Therapeutics

Selective Delivery of PEGylated Compounds toTumor Cells by Anti-PEG Hybrid AntibodiesHsin-Yi Tung1,2,3, Yu-Cheng Su2, Bing-Mae Chen2, Pierre-Alain Burnouf1,2,3,Wei-Chiao Huang2, Kuo-Hsiang Chuang4, Yu-Ting Yan2, Tian-Lu Cheng5, andSteve R. Roffler2

Abstract

Polyethylene glycol (PEG) is attached to many peptides, pro-teins, liposomes, and nanoparticles to reduce their immunogenic-ity and improve their pharmacokinetic and therapeutic properties.Here, we describe hybrid antibodies that can selectively deliverPEGylated medicines, imaging agents, or nanomedicines to targetcells. Human IgG1 hybrid antibodies aPEG:aHER2 and aPEG:aCD19were shownbyELISA, FACS, andplasmonresonance tobindto both PEG and HER2 receptors on SK-BR-3 breast adenocarci-noma and BT-474 breast ductal carcinoma cells or CD19 receptorson Ramos and Raji Burkitt's lymphoma cells. In addition, aPEG:aHER2 specifically targeted PEGylated proteins, liposomes, andnanoparticles to SK-BR-3 cells that overexpressed HER2, but not

toHER2-negativeMCF-7breast adenocarcinomacells. Endocytosisof PEGylated nanoparticles into SK-BR-3 cells was induced specif-ically by the aPEG:aHER2 hybrid antibody, as observed by con-focal imaging of the accumulation of Qdots inside SK-BR-3 cells.Treatment ofHER2þ SK-BR-3 andBT-474 cancer cellswithaPEG:aHER2 and the clinically used chemotherapeutic agent PEGylatedliposomal doxorubicin for 3 hours enhanced the in vitro effective-ness of PEGylated liposomal doxorubicin by over two orders ofmagnitude.Hybrid anti-PEGantibodiesoffer a versatile andsimplemethod to deliver PEGylated compounds to cellular locations andcan potentially enhance the therapeutic efficacy of PEGylatedmedicines. Mol Cancer Ther; 14(6); 1317–26. �2015 AACR.

IntroductionAttachment of polyethylene glycol (PEG), a hydrophilic,

nontoxic, and nonantigenic biocompatible polymer, to pep-tides and proteins can enhance drug stability (1) and bioavail-ability (2) as well as reduce immunogenicity (3). PEGylation ofpolymeric nanoparticles, liposomes, and micelles can alsogreatly extend their circulation time in the body, enhancetumor accumulation, and decrease systemic toxicity, which inturn can improve therapeutic efficacy (4, 5). PEGylation has,therefore, been widely used to improve the properties ofdiagnostic and therapeutic agents.

Nanocarriers can accumulate in tumors via the enhancedpermeability and retention effect due to leakage of nano-sizedmaterials through the tumor vasculature and defects of lymphaticclearance within tumors (6). In addition to the enhanced serumhalf-life afforded by PEGylation, covalent attachment of targetingligands or antibodies to PEGylated nanocarriers can furtherincrease their uptake into tumor cells via receptor-mediatedendocytosis. For example, PEGylated liposomes covalently linkedto anti-HER2 Fab fragments (fragment antigen binding) are moreeffective than nontargeted liposomes for the treatment of mousebreast cancer xenografts (7), due to increased uptake of theimmunoliposomes in tumor cells (8). However, the processes ofcovalently conjugating antibodies can damage their bindingactivity, alter the physiologic and pharmacodynamic propertiesof nanocarriers, and generate new antigenic epitopes (9). Fur-thermore, covalent attachment of antibodies to nanocarriers canhinder the release of therapeutic payloads inside cells (10),thereby decreasing treatment efficacy. Covalent attachment ofantibodies to nanoparticles can also greatly complicate themanufacturing process.

Bispecific antibodies (BsAb) are antibody derivatives that pos-sess two distinct antigen-binding specificities. BsAbs have beendeveloped to simultaneously block two antigens expressed oncancer cells (11), establish crosslinks to activate effector cells atcancer cells (12), and deliver payloads containing drugs, toxins,and imaging reagents to tumors (13). Here, we describe newbispecific (hybrid) antibodies with dual specificities for PEGy-lated compounds or nanocarriers and tumor antigens (Fig. 1A).We show that a humanized hybrid antibody in an intact IgG1

format was able to tether PEGylated compounds to HER2þ cellsand increase the in vitro cytotoxicity of liposomal doxorubicin toHER2þ cancer cells.

1Taiwan International Graduate Program in Molecular Medicine,National Yang-Ming University and Academia Sinica, Taipei, Taiwan.2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.3Institute of Biochemistry and Molecular Biology, National Yang-MingUniversity, Taipei, Taiwan. 4Graduate Institute of Pharmacognosy, Tai-pei Medical University, Taipei, Taiwan. 5Faculty of Biomedical Scienceand Environmental Biology, MedicoGenomic Research Center, Kaoh-siung Medical University, Kaohsiung, Taiwan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Steve R. Roffler, Institute of Biomedical Sciences,Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan. Phone:886-2-2652-3079; Fax: 886-2-2782-9142; E-mail: [email protected]; andTian-Lu Cheng, Department of Biomedical and Environmental Biology, Kaoh-siung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan. Phone:886-7-3121101-2360; Fax: 886-7-3227508; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0151

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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Materials and MethodsReagents

CH3O-PEG1K-NH2, CH3O-PEG2K-NH2, NH2-PEG3K-NH2, OH-PEG5K-NH2, CH3O-PEG5K-NH2, and CH3O-PEG20K-NH2 werepurchased from Sigma-Aldrich. PEGASYS (PEG–IFN-alfa-2a) wasfrom Roche and Neulasta (PEGylated granulocyte-colony stimu-lating factor, G-CSF) was a gift from Amgen. Qtracker655(Qdot655), a nanocrystal semiconductor coated with multiplelinear CH3O-PEG2K molecules, was purchased from Invitrogen.Alexa Fluor 647-N-hydroxysuccinimide ester (NHS ester) waspurchased from Molecular Probes. Doxisome (PEGylated lipo-somal doxorubicin) was from Taiwan Liposome Company,Ltd. Recombinant HER2 extracellular domain (ECD) was a giftfrom Dr. An-Suei Yang (Genomics Research Center, AcademiaSinica, Taiwan). Anti-HER2 antibody (Herceptin) used for detect-ing surface HER2 expression on various cell lines via FACSanalysis was from Roche. Distearoyl phosphatidylcholine (DSPC)and distearoyl phosphatidylethanolamine-PEG2K (DSPE-PEG2K)were purchased from Avanti Polar Lipids.

Cell lines and cultureSK-BR-3 human breast adenocarcinoma (ATCC; HTB-30),

MCF-7 human breast adenocarcinoma (ATCC; HTB-22), JurkatE6-1 human acute T lymphoma cells (ATCC; TIB-152), Rajihuman Burkitt's lymphoma cells (ATCC; CCL-86), HT29 humancolon colorectal adenocarcinoma cells (ADCC;HTB-38), HCC-36human hepatocellular carcinoma cells, and SW-480 (ATCC; CCL-228) human colon colorectal adenocarcinoma cell lines wereobtained from the American Tissue Culture Collection in 1999except for SK-BR-3, which was obtained in 2004. Ramos humanBurkitt's lymphoma cells (ATCC; CRL-1596) and BT-474 humanductal carcinoma (ATCC; HTB-20) cells were obtained from theFood Industry Research and Development Institute (Hsinchu,Taiwan) in 2005 and 2009, respectively. E11, 3.3 and 6.3 mouseanti-PEG hybridoma cells were developed in our laboratory(14, 15). Viral transformed human embryonal kidney 293 FTcells were kindly provided by Dr. Ming-Zong Lai (Institute ofMolecular Biology, Academia Sinica, Taiwan) in 2012. All celllineswereMycoplasma free and cultured according to the suppliers'recommendations. Hybridoma cells, 293 FT cells, and HCC-36cells were cultured in DMEM (Sigma-Aldrich) supplementedwith 6 g/L HEPES, 3.7 g/L NaHCO3, 10% heat-inactivated FBS(HyClone), penicillin (100 U/mL), and streptomycin (100 mg/mL). SK-BR-3, MCF-7, BT-474, Jurkat, Raji, Ramos, HT29, andSW-480 cells were cultured in RPMI-1640 (Life Technologies)supplemented with 6 g/L HEPES, 2 g/L NaHCO3, 10% heat-inactivated FBS (HyClone), penicillin (100 U/mL), and strepto-mycin (100 mg/mL). All cells were cultured at 37�C in a humid-ified atmosphere of 5% CO2 in air. No authentication besidesconfirming surface expression levels of CD19 or HER2 wasperformed by the authors.

Hybrid antibodiesA schematic of hybrid antibody expression constructs is shown

in Fig. 1B. The VH-CH1 and VL-Ck fragments of mouse anti-PEGbackbone antibody 6.3 were amplified from the cDNA isolatedfrom 6.3 hybridoma cells and humanized as described (16). Tofacilitate heterodimer formation of the hybrid antibodies, muta-tions were introduced by site-directed mutagenesis in the humanIgG1 CH3 domain (S354C and T366W) to create a knob structure

and mutations in a separate IgG1 CH3 domain (S349C, T366S,L368A, and Y407V) to create a hole structure (17, 18). Thehumanized 6.3 VH-CH1 domain, assembled with the humanIgG1 Fc domain with knob mutations via assembly PCR (19),was cloned into the pLNCX-hbG-e-B7 vector (20) to acquire thehuman Igk leader sequence (Igk LS) at the N terminus of thehumanized 6.3 (h6.3)-knob heavy-chain expression sequence,whereas an HA tag downstream of the Igk LS was removed. Afurin-2A (F2A; ref. 21)–based bicistronic sequence, and thehumanized 6.3 VL-Ck domain with a Igk LS sequence inserted atthe N terminus, were cloned into the pLNCX-Igk LS-h6.3-knobheavy-chain expression vector to form a Igk LS-h6.3-knob heavy-chain F2A-Igk LS-h6.3 light-chain expression cassette. The entireh6.3-knob antibody expression cassette was subsequently clonedinto the lentivirus expression vector pLKOAS3w-hyg (RNAicore; Academia Sinica).

The human anti-HER2 antibody sequence was cloned fromthe pBub-YCMC plasmid kindly provided by Prof. Louis M.Weiner of Fox Chase Cancer Center (Philadelphia, PA; ref. 22).A human anti-CD19 (BU12) antibody sequence (23) was clonedvia assembly PCR. The CH1 and Ck domains of the HER2 and theCD19 antibodies were individually exchanged, to promoteparing of the correct light and heavy chains (24). The VH-Ckdomains of the anti-HER2 or anti-CD19 antibodies, assembledwith the human IgG1 Fc domain with hole structure mutations(S349C, T366S, L368A, and Y407V) via assembly PCR, werecloned into pLNCX-hbG-e-B7 vector (20) to acquire the humanIgk LS and HA tag at the N terminus of the anti–HER2-hole andanti–CD19-hole heavy-chain expression sequence. A F2A bicis-tronic sequence and theVL-CH1domains of the anti-HER2or anti-CD19 antibodies with an Igk LS sequence inserted at the Nterminus, were cloned into the pLNCX-Igk LS-HA-anti–HER2-hole or pLNCX-Igk LS-HA-anti–CD19-hole heavy-chain expres-sion vector. The entire anti–HER2-hole or anti–CD19-hole anti-body expression cassette was cloned into the lentivirus expressionvector pLKOAS3w-pur (RNAi core; Academia Sinica).

Production of recombinant proteinsRecombinant lentivirus particles were packaged by cotrans-

fection of pLKOAS3w-hyg aPEG-knob, and either pLKOAS3w-pur aCD19-hole or pLKOAS3w-pur aHER2-hole, together withpCMVDR8.91 packaging plasmid and pMD.G VSV-G envelopeplasmid (RNAi core, Academia Sinica). 293 FT cells were seed-ed at 15% of confluence in a 6-well plate in DMEM mediumcontaining 10% FBS 24 hours before viral infection. On thenext day, the cells were infected with lentivirus particles expres-sing aPEG-knob and aCD19-hole, or aPEG-knob and aHER2-hole in the presence of 5 mg/mL polybrene (Sigma-Aldrich).Twenty-four hours later, the cells were selected with hygromy-cin (0.5 mg/mL) and puromycin (3 mg/mL) to generate stablecell lines expressing aPEG:aCD19 or aPEG:aHER2 hybridantibodies.

Purification of hybrid antibodiesA total of 5 � 107 of selected 293FT cells stably expressing

hybrid antibodies in 15 mL culture medium (DMEM with 10%low bovine IgG medium, serum was preabsorbed with proteinA Sepharose 4 Fast Flow chromatography; GE Healthcare) werecultured in a CELLine adhere 1,000 two-compartment biore-actor (Integra Biosciences AG). Antibody-containing culture

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medium was harvested every week. The pooled supernatantwas centrifuged at 910� g for 15 minutes at 4�C to remove cellsand subsequently centrifuged at 21600 � g for 30 minutes at4�C to remove cell debris. The affinity purification process issummarized in Supplementary Fig. S1. Briefly, the clarifiedsupernatant was passed through a G25 column (GE Health-care) in PBS, and the hybrid antibodies were affinity purifiedby protein A Sepharose 4 Fast Flow chromatography (GEHealthcare) and eluted in 0.1 mol/L glycine buffer (pH 3.5)to remove mismatched or excess light chains. The elutedproducts were further purified by cyanogen bromide (CNBr)-CH3O-PEG1K-NH2 affinity chromatography (35 mg of CH3O-PEG1K-NH2 conjugated per gram of CNBr activated Sepharose4B) using PBS-T (0.05% Tween-20) as the elution buffer toremove aHER2-hole or aCD19-hole homodimers. Subse-quently, the eluted hybrid antibodies were purified by affinitychromatography using Pierce anti-HA agarose (Thermo Scien-tific) and 3 mol/L SCN as the elution buffer to remove aPEG-knob homodimers. Purified hybrid antibody fractions werepooled and dialyzed against PBS three times after each affinitypurification step, concentrated using Amicon Ultra (30 kDcutoff; Millipore) and sterile filtered.

Mass spectrometryThe molecular weight of intact hybrid antibodies was deter-

mined on a Voyager DE-STR MALDI-TOF mass spectrometer (ABSciex). 1.5 mg antibody in 0.5 mL water was spotted onto apolished steel target and mixed directly onto the plate with 0.5mL of 45 mmol/L sinapinic acid in 30% acetonitrile/0.3% tri-fluoroacetic acid. The samples were allowed to air dry, and thenloaded into the mass spectrometer. The instrument was operatedin the linear positive ion mode with accelerating voltage of 25 kVand delayed extraction of 1,700 ns to acquire the molecularweight of intact proteins. To improve the signal-to-noise ratio,600 shotswere averaged for eachmass spectrum. An externalmassspectrum calibration was performed using the IgG CalibrationStandard Kit (AB Sciex).

Anti-PEG antibody ELISAMaxisorp 96-well microplates (Nalge-Nunc International)

were coatedwith 10mg/mLCH3O-PEG2K-NH2,NH2-PEG3K-NH2,OH-PEG5K-NH2, or CH3O-PEG20K-NH2 in 50 mL per well0.1 mol/L NaHCO3/Na2CO3 (adjusted to pH 8.0 with HCl)buffer for 3 hours at 37�C and then blocked with 200 mL perwell dilution buffer (5% skim milk in PBS) at 4�C overnight.Graded concentrations of anti-PEG antibodies E11, 3.3 or 6.3 in50 mL 2% skim milk in PBS were added to the plates at roomtemperature (RT) for 1 hour. The plates were washed with PBScontaining 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich) three times. Horse-radish peroxidase (HRP)–conjugated goat anti-human IgG frag-ment crystallizable region (Fc) antibody (Sigma-Aldrich) in 50 mLdilution buffer were used for detecting bound anti-PEG antibo-dies on the microplates. After incubating for 1 hour at RT, theplates were washed as described above. The bound peroxidaseactivity was measured by adding 100 mL per well ABTS solu-tion [0.4 mg/mL, 2,20-azinobis(3-ethylbenzthiazoline-6-sulfonicacid), 0.003%H2O2, and100mmol/Lphosphate–citrate, pH4.0)at RT. The absorbance (405 nm) of wells was measured in amicroplate reader (Molecular Device).

Cell-based ELISATo measure hybrid antibody binding to cancer cells, 96-well

microplates were coated with 2 mg per well poly-L-lysine(40 mg/mL) in PBS for 30 minutes at 37�C, washed twice withPBS and then coated with 2 � 105 SK-BR-3 (HER2þ) or MCF-7(HER2�) cells per well. The cells were centrifuged at 910 � g for5minutes and then fixed with 4%para-formaldehyde for 20min-utes at RT. The plates were washed with PBS four times andblocked with 200 mL per well 2% skimmed milk in PBS at 4�Covernight. The plates were then incubated with graded concen-trations of hybrid antibodies at RT for 1 hour, then washed withPBS-T (0.05% Tween-20) five times and incubated with HRP-conjugated goat anti-human IgG Fc (Sigma-Aldrich) for 1 hourat RT. The plates were washed with PBS-T three times and PBStwice. A 100 mL per well ABTS solution, 0.003% H2O2,100mmol/L phosphate citrate, pH 4.0)was added for 40minutesat RT, and the absorbance of the wells at 405 nm was measuredon a microplate reader.

Flow cytometer analysisTo test the dual binding activity of purified hybrid antibodies

to cancer cells and PEGylated nanoparticles, 2.5 � 105 SK-BR-3(HER2þ), BT-474 (HER2þ) orMCF-7 breast cancer cells (HER2�),CD19� Jurkat T-cell lymphoma cells (CD19�), orRaji (CD19þ) orRamos Burkitt's (CD19þ) lymphoma cells were incubated with10 mg/mL purified hybrid antibodies in RPMI medium for 30minutes at 4�C. After washing with cold 0.05% BSA/PBS twice,the cells were incubated with 10 nmol/L Qdot655 in RPMImedium for 30 minutes at 4�C. The cells were washed with cold0.05% BSA/PBS twice. Afterward, the cells were stained withSYTOX Blue cell viability dye (Invitrogen), and the surface fluo-rescence of Qdot655 on 104 viable cells was measured on a LSR II(BD Biosciences). Fluorescence intensities were analyzed withFlowjo (Tree Star Inc.)

PEGylated protein preparationCH3O-PEG2K-succinimidyl propionic acid (SPA; Shearwater)

dissolved in DMSO at 2mg/mL wasmixed with BSA (10mg/mL)or b-glucuronidase (10mg/mL) at amolar ratio of PEG to proteinof 10 for 2 hours at RT to produce PEGylated BSA or PEGylatedb-glucuronidase. One-tenth volume of 1 mol/L glycine solutionwas added to stop the reaction. PEGylated BSAwas purified via gelfiltration using Sephacryl S-300 HR (GE Healthcare) and PEGy-lated b-glucuronidase was purified via ion exchange using DEAESephadex A-50 (Pharmacia).

Liposome preparationFor generation of PEG-liposomes, DSPC and DSPE-PEG2K

and cholesterol were dissolved in chloroform at a 65:5:30molar ratio, respectively. Naked liposomes were prepared usingDSPC:cholesterol at a molar ratio of 70:30. A dried lipid filmwas formed at 65�C by rotary evaporation (B€uchi, RotavaporRII) and rehydrated in Tris buffered saline (50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4) at 65�C to a final lipidconcentration of 10 mg/mL. The liposomal suspension wassubmitted to 5 freeze/thaw cycles using liquid nitrogen fol-lowed by extrusion 11 times at 70�C through 400, 200, and80 nm polycarbonate membranes each using a mini-extruder(Avanti Polar Lipids, Inc.). Final lipid concentrations weremeasure by Bartlett's assay (25).

Delivery of PEGylated Compounds to Cancer Cells

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Cell-based sandwich ELISACells were coated in 96-well microplates as previously

described and fixed. After adding graded concentrations of hybridantibodies in PBS for 1 hour, the plates were washed and thenincubated with fixed concentrations of naked liposomes(1.4 mg/mL lipid), PEG-liposomes (1.4 mg/mL lipid), E.colib-glucuronidase (60 nmol/L), E.coli b-glucuronidase-PEG2K (60nmol/L), BSA (60 nmol/L), BSA-PEG2K (60 nmol/L), PEGASYS(40 nmol/L), Neulasta (20 nmol/L), or Qdot655 (3 nmol/L) for1 hour. After washing with PBS, the plates were further incubatedwith biotinylated 15-2b antibody (anti–methoxy-PEG; ref. 26)for 1 h to detect bound conjugates. The plates were washed withPBS and further incubatedwith streptavidin-HRP (Jackson Immu-noResearch) for 1 hour. The plates were washed with PBS and100 mL per well ABTS solution and 0.003% H2O2 was added for40 minutes at RT. The absorbance of the wells at 405 nm wasmeasured on a microplate reader.

Confocal live cell imagingHER2þ SK-BR-3 breast cancer cell cells (2 � 105) and HER2�

MCF-7 control cells (2 � 105) cultured in RPMI medium supple-mented with 10% FBSwere seeded on a glass slide precoated with2 mg per well poly-L-lysine (40 mg/mL) overnight. The cells wereincubated with 1 mg/mL Hoechst 33342 (Invitrogen) to stainnuclei and 0.1 mmol/L LysoTracker green DND-26 in RPMI(Sigma-Aldrich) to stain lysosomes for 40 minutes at 37�C in anatmosphere of air containing 5% CO2. After washing with RPMIgrowth medium supplemented with 10% heat-inactivated FBS,the cells were stained with 10 mg/mL aPEG:aHER2 or aPEG:aCD19 hybrid antibodies in 500 mL RPMI growth medium sup-plemented with 10% heat-inactivated FBS for 30 minutes. Afterwashing the cells, 10 nmol/L Qdot655 in 500 mL RPMI growthmedium was added to the cells and further incubated for 5 hoursat 37�C before the fluorescence signals were detected on a ZeissLSM780 laser-scanning microscope (Carl Zeiss AG).

3H-thymidine incorporation assay104 SK-BR3 (HER2þ), BT-474 (HER2þ) or 5 � 103 MCF-7

(HER2�) cells were seeded in 96-well plates overnight. The nextday, 2 or 10 mg/mL aPEG:aHER2 or 10 mg/mL control aPEG:aCD19 hybrid antibodies and graded concentrations of doxisomewere added in triplicate to the wells for 3 hours at 37�C. Afterremoving the medium and adding fresh medium, the cells wereincubated for 48 hours and then pulsed with 3H-thymidine(1mCi/well, specific activity 25.0Ci/mmol;MoravekBiochemicals)for 18 hours. The cells were harvested and the radioactivity wasmeasured on a TopCount microplate scintillation counter (Pack-ard). Results are expressed as the percentage of 3H-thymidineincorporation compared with untreated cells by the followingformula:

The percentage of 3H-thymidine incorporation ¼ 100 � (aver-age sample 3H-thymidine cpm/average untreated control 3H-thymidine cpm). IC50 values were calculated by fitting the datato a log (inhibitor) versus responses (variable slopes model) withPrism 5 software (GraphPad Software).

Statistical analysisStatistical significance of differences between mean values was

estimated with Excel (Microsoft) using the independent Student ttest for unequal variances. P values <0.05 were considered statis-tically significant.

ResultsAnti-PEG monoclonal antibody 6.3 binds PEG with highaffinity

We have generated several hybridoma cells (E11, 3.3 and 6.3)that secrete IgG monoclonal antibodies with specificity for therepeating ethylene oxide subunits in the PEG backbone (15, 27).The anti-PEG antibodies were compared for binding to amino-PEG coated in 96-well ELISA plates (SupplementaryMethods andSupplementary Fig. S2). Amino-PEG was used because the ter-minal amine group allows stable attachment of the PEG mole-cules to the microplates. 6.3 bound to wells coated with PEG5K

with an apparent affinity that was 14-fold greater than 3.3, and34-fold greater than E11 anti-PEG antibodies (SupplementaryTable S1). Likewise, 6.3 displayed superior binding to shortmethylated PEG (CH3O-PEG2K) or long methylated PEG(CH3O-PEG20K) molecules as compared with 3.3 and E11 anti-bodies (Supplementary Fig. S2B and S2C). We therefore choseto use 6.3 to construct hybrid antibodies due to the higher affi-nity of 6.3 as compared with our previous best IgG anti-PEGantibody 3.3 (15).

Expression and purification of hybrid antibodiesWe developed two hybrid antibodies: aPEG:aHER2, which

targets the well-known tumor antigen HER2/neu that is over-expressed in 20% to 30% of human breast and other adenocarci-nomas and aPEG:aCD19, which targets CD19, a surface core-ceptor overexpressed in the majority of B cell tumors. Recombi-nant DNA technology was used to create hybrid antibodiesderived from the cDNA coding regions of the VH and VL domainof either humanized anti-HER2 or anti-CD19 antibodies, andhumanized 6.3 anti-PEGmonoclonal antibody using the "knobs-into-holes" strategy (17) and the immunoglobulin domain cross-over approach (24) for heterodimer formation and correct anti-body heavy- and light-chain assembly. The construction map ofaPEG:aCD19 and aPEG:aHER2 hybrid antibodies is shownin Fig. 1B. The heavy-chain and light-chain domains of both halfantibodies were linked with a F2A bicistronic sequence andcloned into lentivirus expression vectors. The constructs wereused to produce lentivirus particles, which were subsequentlyused for generation of stable 293 FT producer cells. The secretedhybrid antibodies (aPEG:aCD19 and aPEG:aHER2) in the cul-ture medium were sequentially affinity purified on protein Aresin, CNBr-PEG1K beads and anti-HA Sepharose (SupplementaryFig. S1). SDSPAGE analysis showed that purified hybrid anti-bodies were composed of two heavy chains (�55 kDa) and twolight chains (�25 kDa) under reducing conditions (Fig. 1C). Themolecular weights of aPEG:aHER2 and aPEG:aCD19 hybridantibodies were 153.8 and 152.4 kDa, respectively, as determinedby MALDI-TOF.

Functional characterization of hybrid antibodiesThe purified hybrid antibodies were first analyzed for bind-

ing to different PEG molecules by direct ELISA. aPEG:aHER2and aPEG:aCD19 showed similar binding to ELISA micro-plates coated with methoxy PEG (Fig. 2A), diamine PEG(Fig. 2B), and PEG (Fig. 2C), whereas no binding was observedin wells coated with human fibronectin (Fig. 2D) or BSA(Fig. 2E). These results show that the hybrid antibodiesretained PEG-binding activity.

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FACS analysis using an anti-HER2 (Herceptin) antibody wasused to determine HER2 levels on the surface of live HT29 andSW-480 human colon colorectal adenocarcinoma cells, HCC-36human hepatocellular carcinoma cells, as well as on SK-BR-3, BT-474, and MCF-7 human breast cancer cells (SupplementaryMethods). SK-BR-3 cells expressed the highest levels of HER2,followed by BT-474 cells whereas essentially background levels ofHER2were detected onMCF-7,HT-29, SW-480, andHCC-36 cells(Supplementary Fig. S3). Binding of the hybrid antibodies toselected cancer cells was then examined by cell-based ELISA.aPEG:aHER2 bound to HER2þ SK-BR-3 cells but not toHER2� MCF-7 cells (Fig. 3A). As expected, aPEG:aCD19 did notbind to either SK-BR-3 or MCF-7 cells (Fig. 3A). These resultsindicate that aPEG:aHER2 could selectively bind to cells thatexpress HER2 on their surface.

The affinity of aPEG:aHER2 to HER2 ECD was determined byplasmon resonance measurement in a Biacore T200 (GE Health-care; Supplementary Methods). Different concentrations ofaPEG:aHER2 (8 nmol/L to 128 nmol/L) were flowed throughthe cell on a CM5 chip immobilizedwithHER2 ECDproteins andplasmon resonance was measured. aPEG:aHER2 displayed adissociation constant (KD) value of approximately 5 � 10�9

mol/L for binding to HER2 (Supplementary Table S2). Thebinding affinities of the hybrid antibodies to PEGmolecules weredeterminedbymicroscale thermophoresis (SupplementaryMeth-ods). aPEG:aHER2 and aPEG:aCD19 displayed similar bindingaffinity to fluorescently labeled CH3O-PEG5K with KD values ofapproximately 17 � 10�9 and 30 � 10�9 mol/L, respectively(Supplementary Table S2).

Specific delivery of PEGylated nanoparticles (Qdot655) to livecancer cells mediated by the hybrid antibodies was examined byFACS analysis. aPEG:aHER2 mediated strong binding ofQdot655 to HER2þ SK-BR-3 and BT-474 breast cancer cells, butnot to the HER2� MCF-7 breast cancer cells or Ramos and RajiBurkitt's lymphoma cells (Fig. 3B, solid lines). On the other hand,aPEG:aCD19 promoted the binding of Qdot655 to CD19þ

Ramos and Raji cells but not to the CD19� cells (Fig. 3B, dashedlines). We conclude that the hybrid antibodies can selectivelytarget a PEGylated nanoparticle to cells that express the corre-sponding antigen on their surface.

The ability of aPEG:aHER2 to mediate delivery of variousPEGylated compounds to cells that expresses HER2 was investi-gated by cell-based sandwich ELISA. aPEG:aHER2 could tetherPEGylated proteins and liposomes to HER2þ SK-BR-3 cells(Fig. 3C). Binding required PEG because naked liposomes orproteins were not retained atHER2þ cells (Fig. 3C).aPEG:aHER2also mediated binding of commercially available PEGylatednanoparticles (Qdot655) and medicines (PEGASYS and Neu-lasta) to SK-BR-3 cells (Fig. 3D). By contrast, aPEG:aCD19 didnot promote the binding of PEGylated compounds to HER2þ SK-BR3 cells (Fig. 3E) and aPEG:aHER2 did not mediate binding ofPEGylated compounds to HER2� MCF-7 cells (Fig. 3F). Takentogether, these results demonstrate that aPEG:aHER2 couldmediate specific delivery of diverse PEGylated entities to cancercells that express HER2 on their surface.

aPEG:aHER2 can induce endocytosis of PEGylatednanoparticles into HER2þ cancer cells

We investigated whether aPEG:aHER2 could promote theinternalization of Qdot655 PEGylated nanoparticles into tumorcells. Confocal microscopy demonstrated that aPEG:aHER2

Figure 1.Illustration of the dual-binding function of hybrid antibodies, construction,expression, and purification of hybrid antibodies. A, illustration of selectivedelivery of PEGylated compounds to HER2þ cancer cells by an anti-PEGhybrid antibody. The hybrid IgG antibody possesses two antigen-bindingsites, one for PEG and one for HER2 on cancer cells. The hybrid antibody alsopossesses an Fc domain that can potentially bind to Fc receptors on accessoryimmune cells. The PEGylated compounds represent PEGylated DNA,PEGylated peptide, PEGylated protein, and PEG-liposome (left to right). B,schematic of hybrid antibody expression constructs. aPEG-knob codes for ahuman Igk signal peptide, the humanized VH domain of anti-PEG antibody 6.3,a human IgG1 heavy chain with knob mutations (S354C, T366W), followed bya F2A bicistronic sequence, the humanized VL domain of 6.3 and a human Ck

domain. aCD19-hole and aHER2-hole code for a human Igk signal peptide, aHA tag, the humanized VH domain of an anti-CD19 or anti-HER2 antibody, ahuman Ck domain, a human IgG1 heavy-chain Fc domain with hole mutations(S349C, T366S, L368A, and Y407V), followed by a F2A bicistronic sequence, ahuman Igk signal peptide, the humanized VL domain of the anti-CD19 or anti-HER2 antibody and a human IgG1 CH1 domain. CH, heavy-chain constantdomain; VH, heavy chain variable domain; VL, light chain variable domain; Ck, klight chain constant domain; LS, leader sequence; HA, human influenzahemagglutinin epitope tag. C, reducing SDSPAGE showing the purified hybridantibodies and half antibodies separately expressed by different 293 FT cellclones; H, heavy chain; L, light chain.






could specifically deliver Qdot655 to SK-BR-3 cells, accompaniedwith accumulation of Qdot655 inside cells after 5 hours ofincubation at 37�C (Fig. 4A, top). By contrast, the uptake ofQdot655 was not observed in SK-BR-3 cells incubated withcontrol aPEG:aCD19 hybrid antibody and Qdot655 (Fig. 4A,

bottom). Binding and uptake of Qdot655 was also not observedin HER2� MCF-7 cells treated with aPEG:aHER2 (Fig. 4B). Weconclude that aPEG:aHER2 can mediate selective bindingand internalization of PEGylated nanoparticles into HER2þ can-cer cells.

Figure 2.ELISA analysis of hybrid antibodybinding to PEGmolecules. The bindingof aPEG:aCD19 or aPEG:aHER2hybrid antibodies to plates precoatedwith CH3O-PEG2K-NH2 (A),NH2-PEG3K-NH2 (B), OH-PEG5K-NH2

(C), human fibronectin (D), or BSA (E)were measured by direct ELISA; mean� SD; n ¼ 3.

Figure 3.Hybrid antibodies can targetPEGylated compounds to cancer cells.A, binding of hybrid antibodies to SK-BR-3 and MCF-7 cells was determinedby direct ELISA; n ¼ 2; bars, SD. B,FACS analysis of the dual binding ofaPEG:aHER2 (solid lines) or aPEG:aCD19 (dashed lines) hybrid antibodiesto cancer cells and PEGylatedQdot655. Shaded curves show theFACs of unstained cells. C, ELISAmeasurement of the binding of theindicated compounds to SK-BR-3 cellsmediated by aPEG:aHER2. D, ELISAmeasurement of the binding ofcommercial PEGylated compounds toSK-BR-3 cells mediated by aPEG:aHER2. E, ELISA measurement of thebinding of commercial PEGylatedcompounds to SK-BR-3 cells mediatedby aPEG:aCD19. F, ELISAmeasurement of the binding ofcompounds to MCF-7 cells mediatedby aPEG:aHER2; mean � SD; n ¼ 3.

Tung et al.





aPEG:aHER2 can enhance the cytotoxicity of doxisome toHER2þ cancer cells

BecauseaPEG:aHER2 triggered the endocytosis of a PEGylatednanoparticle in HER2þ cancer cells, we investigated whether itcould enhance the cytotoxicity of PEGylated liposomal doxoru-bicin (doxisome) in cancer cells. After 3 hours exposure of thecells to hybrid antibodies and doxisome or to doxisome alone,the cells were refreshed with new medium and cultured for48 hours to allow time for the doxorubicin to kill the cancercells. We then assessed the proliferation of the remaining cells byadding 3H-thymidine for 18 hours and measuring the incorpo-ration of radiolabeled 3H-thymidine in newly synthesized DNA.

This assay measures cell proliferation and gives similar results asthe tetrazolium MTT assay (28, 29). aPEG:aHER2 but notaPEG:aCD19 significantly enhanced the cytotoxicity of doxisometo SK-BR-3 (Fig. 5A) and BT-474 (Fig. 5B) HER2þ cancer cells ascompared with treatment with doxisome alone. The similarcytotoxicity observed with doxisome and both 10 and 2 mg/mLaPEG:aHER2 suggests that sufficient hybrid antibodywas presentto saturate theHER2 receptors on SK-BR-3 cells (Fig. 5A). The IC50

values of doxisome in combination with aPEG:aHER2 wereover two orders of magnitude lower than with doxisome alone(Table 1). Neither aPEG:aHER2 or aPEG:aCD19 altered thesensitivity of HER2� MCF-7 cells to doxisome (Fig. 5C), showing

Figure 4.aPEG:aHER2 can induce the bindingand internalization of Qdot655 intoHER2þ cancer cells. SK-BR-3(HER2þ; A) cells and MCF-7 (HER2�;B) cells were incubated with Hoechst33342 (blue) and LysoTracker GreenDND-26 (green) before addition ofhybrid antibodies on ice for 30minutes. Qdot655 (red) were addedand the cells were cultured for 5 hoursat 37�C before confocal images weretaken.

Figure 5.Enhancement of doxisomecytotoxicity by hybrid antibodies.Proliferation of SK-BR-3 (A), BT-474(B), and MCF-7 (C) cells treated withdoxisome alone or doxisomecombined with aPEG:aHER2 orcontrol aPEG:aCD19 hybridantibodies. D, proliferation of SK-BR-3and BT-474 cells incubated withhybrid antibodies or medium; mean �SD; n ¼ 3.






that the hybrid antibodies displayed specificity for cells thatexpressed target ligand. The hybrid antibodies alone did notinduce cytotoxicity in SK-BR-3 and BT-474 cells (Fig. 5D).

DiscussionWe developed and investigated hybrid antibodies consisting of

PEG-binding and tumor antigen-binding half antibodies. Thehybrid antibodies are capable of delivering diverse PEGylatedcompounds and nanoparticles to cancer cells that express surfaceantigens recognized by the tumor antigen-binding half antibody.We found that a aPEG:aHER2 hybrid antibody facilitated theuptake of nanoparticles into HER2þ cancer cells and greatlyenhanced the cytotoxicity of doxisome to HER2þ cancer cells.We anticipate that anti-PEG hybrid antibodies may serve as aversatile approach for targeted delivery of PEGylated compounds(therapeutic proteins, liposomal drugs, gene delivery carriers, orimaging agents) to tumors and other cellular locations, and mayfurther enhance therapeutic and diagnostic efficacy due to theenhanced cellular uptake of PEGylated compounds.

We previously described anti-PEG IgG antibodies (3.3 andE11) with well-characterized binding to PEG (14, 15, 30). How-ever, we were unable to achieve good tumor targeting using E11or 3.3 anti-PEG antibodies, supposedly due to insufficient bind-ing affinity to PEG (unpublished results). We therefore developedanewanti-PEGmonoclonal antibody, 6.3,whichdisplayed aboutone order ofmagnitude higher apparent affinity to PEGmoleculesas compared with our previous high-affinity anti-PEG IgGmono-clonal antibody 3.3 (Supplementary Fig. S2 and SupplementaryTable S1). These results suggest that high affinity is required for theanti-PEG arm of the hybrid antibodies for good delivery ofPEGylated compounds to cellular targets.

Although PEGylation can extend the half-lives and stabilityof nanoparticles, the steric hindrance provided by PEG molec-ules can interfere with desired interactions with target cells. Forexample, PEG molecules on nanocarriers designed for deliveryof DNA or siRNA were found to hamper cellular uptake (31),which is known as the "PEG dilemma" (32). DNA or siRNAmustbe transported across the cell plasma membrane to enter cells,but the interactions between the gene carriers and cell surface areblocked by the aqueous phase surrounding the PEG moieties.Therefore, although PEGylation can extend the stability andhalf-lives of nanoparticles, a trade-off exists as the interactionsbetween the PEGylated compounds and their targets might alsobe hampered by PEGylation.

Active targeting has been one approach to enhance cellularuptake of PEGylated compounds. Target ligands recognizingreceptors specifically overexpressed on cancer cells have beencovalently linked to PEGylated nanoparticles, which in turnpromote selective binding and uptake of the nanoparticles bytarget cells. Proteins, peptides containing specific motifs such asarginine-glycine-aspartic acid (RGD; ref. 33), folate (34), anti-

bodies and antibody fragments (immunoliposomes), nucleicacids (35), and transferrin (36) have been conjugated to PEGy-lated nanoparticles to facilitate active targeting.

Anti-PEG hybrid antibodies may possess some advantagescompared with other targeting technologies. Chemical conjuga-tion is complicated by potential damage to the targeting ligandand cargo (10, 37) as well as by generation of immunogenicepitopes. Interactions between cellular receptors and conjugatedligands can also be hampered by the steric hindrance provided byPEG moieties (38). By contrast, well-characterized hybrid anti-bodies specific to PEGmolecules and cellular receptors may exertconsistent binding of both target cells and PEGylated compoundsunder physiologic conditions, omitting complexities resultedfrom conjugation reactions. Moreover, payloads noncovalentlybound by antibodies may be amore efficient alternative for cargorelease, as targeting ligands may require degradation or compli-cated cleavage processes to release the active cargos (10). Impor-tantly, separation of antibody development and nanoparticles ordrug development may allow for simpler manufacturing process-es. Other BsAbs have been developed to deliver nanocargos tocellular targets. For example, BsAbs recognizing both a chemicalhapten and a tumor antigen were shown to successfully targethapten-conjugated vehicles to specific cells (13, 39). However,chemical conjugation of haptens to the payloads is required.PEGylation of hapten-conjugated nanoparticles can also hinderbinding of anti-hapten BsAbs (40). We overcame this limitationby creating BsAbs that directly bind to PEG molecules alreadypresent on many nanaomedicines.

In contrast with our previously described anti–methoxy-PEGBsAb fragments (26), here, we investigated intact IgG1 hybridantibodies that bind to the ethylene oxide backbone of PEG. Onepotential advantage of human IgG1 hybrid antibodies is theopportunity for both targeted delivery of therapeutic agents andinduction of anticancer immune responses. The Fc domain ofhuman IgG1when bound to the surface of cancer cells can activatecomplement and trigger antibody-dependent cellular cytotoxicity(ADCC) by immune effector cells (natural killer cells and mono-cytes), which contribute to effective antibody therapies (41). Itwill be of interest to determine whether there are any synergisticeffects between ADCC and hybrid antibody delivery of nanome-dicines. Immunostimulatory PEGylated cytokines such as IL2,IFNa, IFNg , and IL12 (42) may also represent rational targets forselective tumor delivery by anti-PEG hybrid antibodies.

In conclusion, our study suggests that anti-PEG hybrid anti-bodies offer a simple and versatile method to selectively deliverPEGylated compounds the target cells. Examples of clinically usedPEGylated proteins include PEG-asparaginase (43), PEG40K

–IFNa 2a (PEGASYS; ref. 44). PEG12K–IFNa 2b (PEG-Intron;ref. 45), PEG20K–filgrastim (Neulasta; ref. 46), and PEGylatedepoietin (47). PEGylated nanomedicines include such examplesas polyplex, a nanoparticle inhalation gene delivery system forgene therapy (48), stealth radiolabeled PEG2K-liposomes and

Table 1. Cytotoxicity of doxisome or doxisome combined with 10 mg/mL aPEG:aHER2 or aPEG:aCD19 hybrid antibodies

Cells Doxisome(mmol/L)

aPEG:aHER2 þ doxisome(mmol/L)

aPEG:aCD19 þ doxisome(mmol/L)

Foldenhancementa

SK-BR-3 34.0 � 8.0 0.07 � 0.0�� 20.6 � 1.7 519 � 176�

BT-474 14.0 � 0.2 0.02 � 0.0�� 10.8 � 1.5 715 � 173��

NOTE: Concentrations of doxisome that produce 50% inhibition of 3H-thymidine incorporation into cellular DNA are indicated in mmol/L (IC50). Results representmean values of triplicate determinations � SD. Significant differences relative to doxisome are indicated; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.aFold enhancement of aPEG:aHER2 þ doxisome versus doxisome alone.

Tung et al.





PEG2K-liposomes encapsulating drugs, including Doxil (49) andDaunoXome (50), and other PEGylated compounds, includingQdot (51), PEG-aptamer pegaptanib sodium (Macugen, Pfizer),PEG small organic molecules and PEG-antibody fragmentsertolizumab pegol (Cimzia, UCB; refs. 5, 52). These and newPEGylated drugs, nanomedicines, and imaging agents underdevelopment represent potential sources for targeted delivery byhybrid anti-PEG antibodies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H.-Y. Tung, Y.-C. Su, T.-L. Cheng, S.R. RofflerDevelopment ofmethodology:H.-Y. Tung, Y.-C. Su, K.-H.Chuang, T.-L. Cheng,S.R. RofflerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.-Y. Tung, B.-M. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.-Y. Tung, Y.-C. SuWriting, review, and/or revision of the manuscript: H.-Y. Tung, Y.-T. Yan,S.R. RofflerAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): H.-Y. Tung, P.-A. Burnouf, W.-C. Huang,T.-L. ChengStudy supervision: Y.-T. Yan, S.R. Roffler

AcknowledgmentsThe authors thank Dr. Shu-Chuan Jao of the Biophysics Core Facility,

Scientific Instrument Center at Academia Sinica for assistance in operatingthe Biacore T200 and Monolith NT.115 pico instruments. Also, the authorsthank Chia-Chen Tai and Tzu-Wen Tai of the Flow Cytometry Core, ScientificInstrument Center at the Institute of Biomedical Sciences, Academia Sinica,Taipei, Taiwan for assistance with the LSR II flow cytometer. In addition, theauthors thank the Protein Mass Core, Scientific Instrument Center at theInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, for anti-body molecular weight measurements. The authors appreciate the help fromthe National RNAi Core Facility, Institute of Molecular Biology/GenomicResearch Center, at Academia Sinica, Taipei, Taiwan for producing recom-binant lentiviruses. The authors especially appreciate help from Dr. An-SueiYang of the Genomic Research Center at Academia Sinica for providingHER2 ECD protein.

Grant SupportThis work was supported by a grant from the Academia Sinica Research

Program on Nanoscience and Nanotechnology (to S.R. Roffler).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 16, 2015; revised March 23, 2015; accepted March 25,2015; published OnlineFirst April 7, 2015.

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2015;14:1317-1326. Published OnlineFirst April 7, 2015.Mol Cancer Ther Hsin-Yi Tung, Yu-Cheng Su, Bing-Mae Chen, et al. Anti-PEG Hybrid AntibodiesSelective Delivery of PEGylated Compounds to Tumor Cells by

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